Enzymatic Synthesis of Deoxyribonucleotides

X. REDUCTION OF PURINE RIBONUCLEOTIDES; ALLOSTERIC BEHAVIOR AND SUBSTRATE SPECIFICITY OF THE ENZYME SYSTEM FROM ESCHERICHIA COLI B

From the ¹ From the Department of Chemistry II, Karolinska Institutet, Stockholm, Sweden

The ribonucleoside diphosphate reductase system from Escherichia coli B, which catalyzes the reductive formation of deoxycytidine diphosphate and deoxyuridine diphosphate from CDP and UDP, also catalyzed the corresponding reductions of adenosine diphosphate and guanosine diphosphate. Evidence was obtained that the identical highly purified proteins (Enzymes B1 and B2, thioredoxin and thioredoxin reductase) participated in all four reactions. Under optimal conditions the maximal velocity with each substrate was of about the same magnitude.

The specificity of the enzyme system was strongly influenced by different nucleoside triphosphates. ATP, which stimulated the reductions of pyrimidine ribonucleotides, had little effect on the reductions of purine ribonucleotides. Rather, these reactions were stimulated by deoxythymidine triphosphate and dGTP, dTTP being the somewhat better stimulator for GDP reduction, and dGTP for ADP reduction. dATP strongly inhibited both reactions, and this inhibition was reversed by ATP but not by dTTP.

According to our present interpretation the different nucleotides act as allosteric effectors, stabilizing certain states of one or several of the enzymes and thereby determining the substrate specificity. Thus ATP stabilizes a state favorable for the reduction of pyrimidine ribonucleotides; dGTP, a state favorable for purine ribonucleotides; and dTTP, a state favorable for both types of nucleotides. dATP stabilizes an inactive state of the enzyme. The simultaneous presence of ATP and dTTP results in a relative inhibition which is much more pronounced with pyrimidine ribonucleotides than with purine ribonucleotides.

These effects may form the basis of a physiological mechanism responsible for a balanced supply of the substrates required for the enzymatic synthesis of deoxyribonucleic acid.

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